Fig 2: SCFA-dependent GPR43 signaling confers GI GVHD protection through the ERK–NLRP3 pathway. B6 WT and Gpr43-/- mice received BMT from syngeneic or allogeneic donors. a Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 normalized to the presence of ß-actin in IECs (CD326+) from allogeneic recipients 14 days after BMT (n = 4–5 each, two-tailed Mann–Whitney U test). b Serum IL-18 levels at day 7 and 14 after BMT (n = 4 WT, n = 5 Gpr43-/-, two-tailed unpaired t-test). c Representative immunoblots and densitometric analysis of cleaved caspase 1 and cleaved IL-18 at day 14 after BMT in IECs (CD326+) from allogeneic recipients. Mice were treated with vehicle or propionate (15 mg kg-1 per day) from day 0 (n = 4 WT, n = 3 Gpr43-/-, two-tailed Mann–Whitney U test). d Serum IL-18 levels from allogeneic recipients treated with vehicle or propionate (15 mg kg-1 per day) at day 7 and 14 after BMT (n = 5 WT each, n = 4 Gpr43-/- each, two-tailed unpaired t-test). e Immunohistochemical staining of IL-18 in colon sections from WT B6 and Gpr43-/- recipients 14 days after BMT treated with vehicle or propionate (15 mg kg-1 per day) from day 0 (n = 3–5 each). f IL-22 levels in serum, homogenized tissues from syngeneic and allogeneic recipients at day 14 after BMT (n = 6 WT syn, n = 4 Gpr43-/- syn, n = 5 WT allo, n = 10 Gpr43-/- allo, two-tailed unpaired t-test). g WT B6 and Gpr43-/- mice received oral administrations of vehicle or propionate (15 mg kg-1 per day) for 3 days. Representative immunoblots and densitometric analysis of phosphorylated ERK normalized to total ERK in IECs (CD326+) after treatment (n = 4 WT each, n = 3 Gpr43-/- each, two-tailed Mann–Whitney U test). h Colon explants from WT B6 mice were pretreated with U0126 (10 µM) for 30 min. Then tissues were primed with LPS (0.1 µg per ml) for 2 h and activated with butyrate or propionate (1 mM) for 2 h. Supernatants were assayed for IL-18 by ELISA (n = 3 each, two-tailed unpaired t-test). Data are representative of two to three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, error bars show the mean ± s.e.m.

Fig 3: a HER2-staining by IHC in the gastric cancer cell lines (i) ESO26, (ii) NCI-N87, (iii) OE19, (iv) SNU16 and (v) KATOIII. Images i, ii and iii were taken at 10X magnification, while images iv, and v were taken at 20X magnification. b Western blot analysis of expression of p-AKT, AKT, p-P70S6K (T389), P70S6K, MAPK ERK 1/2 (T202/Y204), MAPK ERK 1/2, PI3K P110a, and ß-actin in the gastric cancer cell lines NCI-N87, ESO26, OE19, KATOIII and SNU16. All gels were run under the same experimental conditions and the experiments were repeated three times. Representative images are shown

Fig 4: Possible model for the role of the disulfide bridges in the transmembrane region of Torso.In wild-type Torso (upper case), PTTH binding to the extracellular region triggers phosphorylation in the intracellular region by its own kinase activity, and the autophosphorylation facilitates ERK phosphorylation in the downstream signaling pathway. In contrast, without the disulfide bridges in the transmembrane region, as in the phenylalanine mutant (C381/393/394F) (lower case), Torso spontaneously forms a non-covalently-associated dimer, rather than a disulfide-bond-mediated dimer, on the cellular membrane even in the absence of the ligand, and the dimerization causes the ligand-independent receptor autophosphorylation. Although the phosphorylation sites were not identified in this study, the sites in the unregulated, ligand-independent autophosphorylation of the receptor lacking the intermolecular linkage may be different from those in the ligand-dependent phosphorylation of the wild-type Torso, because the unregulated autophosphorylation cannot facilitate downstream ERK phosphorylation. The intermolecular disulfide bridges in the transmembrane region maintain the normal ligand-dependent receptor functions, by preserving the proper relative positioning of the two Torso molecules.

Fig 5: MiR-132 expression is MAPK independent while CREB- and salt inducible kinase-dependent.a, b p38 inhibitor SB-203580 inhibits p38 activity (a), and ERK inhibitor PD-98059 inhibits ERK1/2 activity (b), as demonstrated by western blot for total p38 and ERK1/2, as well as phosphorylated p38 (p-p38) and phosphorylated ERK (p-ERK1/2). GAPDH is used as household gene, representative images of n = 3. c low salt-induced Cox-2 gene expression is inhibited by p38 and ERK inhibition after 24 h, as determined by RT-qPCR. Cox-2 expression is normalized to Gapdh. d p38 and ERK inhibition does not affect miR-132 expression after 2 or 24 h under low salt conditions. e CREB activator Forskolin induces miR-132 expression after 24 h. f CREB-inhibitor 666-15 reduces low salt induced miR-132 expression. g Salt inducible kinase inhibitor MRT67307 increases high-salt-reduced miR-132 expression. d–g As determined by RT-qPCR. Mir-132 is normalized to U6. For panels c–g: n = 3 (averages of independent experiments) (h) working hypothesis of salt dependent miR-132 expression. sc scramblemir, a132 antagomir-132, SB p38 inhibitor SB-203580, PD ERK inhibitor PD-98059. *P < 0.05, **P < 0.01, ***P < 0.001, data are represented as mean ± SD.